This patent application claims priority to U.S. patent application Ser. No. 08/540,814, filed Oct. 11, 1995, and U.S. Pat. No. 5,736,330, filed Oct. 11, 1995, and PCT application Ser. No. PCT/US/96/16198, filed Oct. 10, 1996, all of which are incorporated herein by reference, including all reference cited therein.
Microfiche appendix A contains a listing of selected Visual Basic and C programming source code in accordance with the inventive multiplexed assay method. Microfiche appendix A, comprising 1 sheet having a total of 58 frames, contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent disclosure, as it appears in the Patent and Trademark Office patent files or records, but otherwise reserves all copyright rights whatsoever.
The invention relates generally to laboratory diagnostic and genetic analysis and, more particularly, to a flow cytometric method for the simultaneous and multiplexed diagnostic and genetic analysis of clinical specimens.
Analysis of clinical specimens is important in science and medicine. A wide variety of assays to determine qualitative and/or quantitative characteristics of a specimen are known in the art. Detection of multiple analytes, or separately identifiable characteristics of one or more analytes, through single-step assay processes are presently not possible or, to the extent possible, have provided only very limited capability and have not yielded satisfactory results. Some of the reasons for these disappointing results include the extended times typically required to enable the detection and classification of multiple analytes, the inherent limitations of known reagents, the low sensitivities achievable in prior art assays which often lead to significant analytical errors and the unwieldy collection, classification, and analysis of prior art algorithms vis a vis the large amounts of data obtained and the subsequent computational requirements to analyze that data.
Clearly, it would be an improvement in the art to have adequate apparatus and methods for reliably performing real-time multiple determinations, substantially simultaneously, through a single or limited step assay process. A capability to perform simultaneous, multiple determinations in a single assay process is known as xe2x80x9cmultiplexingxe2x80x9d and a process to implement such a capability is a xe2x80x9cmultiplexed assay.xe2x80x9d
Flow Cytometry
One well known prior art technique used in assay procedures for which a multiplexed assay capability would be particularly advantageous is flow cytometry. Flow cytometry is an optical technique that analyzes particular particles in a fluid mixture based on the particles"" optical characteristics using an instrument known as a flow cytometer. Background information on flow cytometry may be found in Shapiro, xe2x80x9cPractical Flow Cytometry,xe2x80x9d Third Ed. (Alan R. Liss, Inc. 1995); and Melamed et al., xe2x80x9cFlow Cytometry and Sorting,xe2x80x9d Second Ed. (Wiley-Liss 1990), which are incorporated herein by reference. a Flow cytometers hydrodynamically focus a fluid suspension of particles into a thin stream so that the particles flow down the stream in substantially single file and pass through an examination zone. A focused light beam, such as a laser beam illuminates the particles as they flow through the examination zone. Optical detectors within the flow cytometer measure certain characteristics of the light as it interacts with the particles. Commonly used flow cytometers such as the Becton-Dickinson Immunocytometry Systems xe2x80x9cFACSCANxe2x80x9d (San Jose, Calif.) can measure forward light scatter (generally correlated with the refractive index and size of the particle being illuminated), side light scatter (generally correlated with the particle""s size), and particle fluorescence at one or more wavelengths. (Fluorescence is typically imparted by incorporating, or attaching a fluorochrome within the particle.) Flow cytometers and various techniques for their use are described in, generally, in xe2x80x9cPractical Flow Cytometryxe2x80x9d by Howard M. Shapiro (Alan R. Liss, Inc., 1985) and xe2x80x9cFlow Cytometry and Sorting, Second Editionxe2x80x9d edited by Melamed et al. (Wiley-Liss, 1990).
One skilled in the art will recognize that one type of xe2x80x9cparticlexe2x80x9d analyzed by a flow cytometer may be man-made microspheres or beads. Microspheres or beads for use in flow cytometry are generally known in the art and may be obtained from manufacturers such as Spherotech (Libertyville, Ill.), and Molecular Probes (Eugene, Oreg.).
Although a multiplexed analysis capability theoretically would provide enormous benefits in the art of flow cytometry, very little multiplexing capability has been previously achieved. Prior multiplexed assays have obtained only a limited number of determinations. A review of some of these prior art techniques is provided by McHugh, xe2x80x9cFlow Microsphere Immunoassay for the Quantitative and Simultaneous Detection of Multiple Soluble Analytes,xe2x80x9d in Methods in Cell Biology, 42, Part B, (Academic Press, 1994). For example, McHugh et al., xe2x80x9cMicrosphere-Based Fluorescence Immunoassays Using Flow Cytometry Instrumentation,xe2x80x9d in Clinical Flow Cytometry Ed. K. D. Bauer, et al., Williams and Williams, Baltimore, Md., 1993, 535-544, describe an assay where microspheres of different sizes are used as supports and the identification of microspheres associated with different analytes was based on distinguishing a microsphere""s size. Other references in this area include Lindmo, et al., xe2x80x9cImmunometric Assay by Flow Cytometry Using Mixtures of Two Particle Types of Different Affinity,xe2x80x9d J. Immun. Meth., 126, 183-189 (1990); McHugh, xe2x80x9cFlow Cytometry and the Application of Microsphere-Based Fluorescence Immunoassays,xe2x80x9d Immunochemica, 5, 116 (1991); Horan et al., xe2x80x9cFluid Phase Particle Fluorescence Analysis: Rheumatoid Factor Specificity Evaluated by Laser Flow Cytophotometryxe2x80x9d in Immunoassays in the Clinical Laboratory, 185-198 (Liss 1979); Wilson et al., xe2x80x9cA New Microsphere-Based Immunofluorescence Assay Using Flow Cytometry,xe2x80x9d J. Immunological Methods, 107, 225-230 (1988); and Fulwyler et al., xe2x80x9cFlow Microsphere Immunoassay for the Quantitative and Simultaneous Detection of Multiple Soluble Analytes,xe2x80x9d Meth. Cell Biol., 33, 613-629 (1990).
The above cited methods have been unsatisfactory as applied to provide a filly multiplexed assay capable of real-time analysis of more than a few different analytes. For example, certain of the assay methods replaced a single ELISA procedure with a flow cytometer-based assay. These methods were based on only a few characteristics of the particles under analysis and enabled simultaneous determination of only a very few analytes in the assay. Also, the analytic determinations made were hampered due to software limitations including the inability to perform real-time processing of the acquired assay data. In summary, although it has been previously hypothesized that flow cytometry may possibly be adapted to operate and provide benefit in a multiple analyte assay process, such an adaptation has not in reality been accomplished.
Analysis of Genetic Information
The availability of genetic information and association of disease with mutation(s) of critical genes has generated a rich field of clinical analysis. In particular, the use polymerase chain reaction (PCR) and its variants have facilitated genetic analysis. A major advance in this field is described in our co-pending and contemporaneously filed U.S. Application entitled xe2x80x9cMethods and Compositions for Flow Cytometric Determination of DNA Sequences.xe2x80x9d This co-pending application describes a powerful flow cytometric assay for PCR products, which may be multiplexed in accordance with the present invention. A multiplexed flow cytometric assay for PCR reaction products would provide a significant advantage in the field of genetic analysis.
Recent advances in genetic analyses have provided a wealth of information regarding specific mutations occurring in particular genes in given disease states. Consequently, use of an individual""s genetic information in diagnosis of disease is becoming increasingly prevalent. Genes responsible for disease have been cloned and characterized in a number of cases, and it has been shown that responsible genetic defects may be a gross gene alteration, a small gene alteration, or even in some cases, a point mutation. There are a number of reported examples of diseases caused by genetic mutations. Testing of gene expression by analysis of cDNA or mRNA, and testing of normal genes and alleles, as in cases of tissue typing and forensics, are becoming ,widespread. Other uses of DNA analysis, for example in paternity testing, etc., are also important and can be used in accordance with the invention.
Current techniques for genetic analysis have been greatly facilitated by the development and use of polymerase chain reaction (PCR) to amplf selected segments of DNA. The power and sensitivity of the PCR has prompted its application to a wide variety of analytical problems in which detection of DNA or RNA sequences is required.
PCR is capable of amplifying short fragments of DNA, providing short (20 bases or more) nucleotides are supplied as primers. The primers anneal to either end of a span of denatured DNA target and, upon renaturation, enzymes synthesize the intervening complementary sequences by extending the primer along the target strand. During denaturation, the temperature is raised to break apart the target and newly synthesized complementary sequence. Upon cooling, renaturation and annealing, primers bind to the target and the newly made opposite strand and now the primer is extended again creating the complement. The result is that in each cycle of heating and renaturation followed by primer extension, the amount of target sequence is doubled.
One major difficulty with adoption of PCR is the cumbersome nature of the methods of analyzing the reaction""s amplified DNA products. Methods for detecting genetic abnormalities and PCR products have been described but they are cumbersome and time consuming. For example, U.S. Pat. No. 5,429,923 issued Jul. 4, 1995 to Seidman, et al., describes a method for detecting mutations in persons having, or suspected of having, hypertrophic cardiomyopathy. That method involves amplifying a DNA sequence suspected of containing the disease associated mutation, combining the amplified product with an RNA probe to produce an RNA-DNA hybrid and detecting the mutation by digesting unhybridized portions of the RNA strand by treating the hybridized product with an RNAse to detect mutations, and then measuring the size of the products of the RNAse reaction to determine whethercleavage of the RNA molecule has occurred.
Other methods used for detecting mutations in DNA sequences, including direct sequencing methods (Maxim and Gilbert, Proc. Natl. Acad. Sci. U.S.A., 74, 560-564, 1977); PCR amplification of specific alleles, PASA (Botttema and Sommer, Muta. Res., 288, 93-102, 1993); and reverse dot blot method (Kawasaki, et al., Methods in Enzymology, 218, 369-81, 1993) have been described. These techniques, while useful, are time consuming and cumbersome and for that reason are not readily adaptable to diagnostic assays for use on a large scale.
At least one use of flow cytometry for the assay of a PCR product has been reported but that assay has not been adapted to multiplexing. See Vlieget et al., xe2x80x9cQuantitation of Polymerase Chain Reaction Products by Hybridization-Based Assays with Fluorescent Colorimetric, or Chemiluminescent Detection,xe2x80x9d Anal. Biochem., 205, 1-7 (1992). In Vlieger et al. a PCR product was labeled using primers that contained biotinylated nucleotides. Unreacted primers were first removed and the amplified portion annealed with a labeled complementary probe in solution. Beaded microspheres of avidin were then attached to the annealed complementary material. The avidin beads bearing the annealed complementary material were then processed by a flow cytometer. The procedure was limited, inter alia, in that avidin beads having only a single specificity were employed. Further, real-time analysis of the assay""s data was not possible.
Data Manipulation
The large volume of data typically generated during flow cytometric multiple analyte assays, combined with the limited capabilities of prior techniques to collect, sort and analyze such data have provided significant obstacles in achieving a satisfactory multiplexed assay. The computing methods used in prior art flow cytometric analyses have generally been insufficient and unsuited for accurately and timely analyzing large volumes of data such as would be generated by multiplexed assays; particularly when more than two analytes (or properties of a single analyte) are to be simultaneously determined.
The present invention enables the simultaneous determination of multiple distinct analytes to a far greater degree than existing techniques. Further, the invention provides an improved data classification and analysis methodology that enables the meaningful analysis of highly multiplexed assays in real-time. The invention is broadly applicable to multiplexed analysis of a number of analytes in a host of bioassays in which there is currently a need in the art.
The present invention provides improved methods, instrumentation, and products for detecting multiple analytes in a fluid sample by flow cytometric analysis and for analyzing and presenting the data in real-time. An advantage of the invention is that it allows one rapidly and simultaneously to detect a wide variety of analytes of interest in a single assay step.
The invention employs a pool of bead subsets. The individual subsets are prepared so that beads within a subset are relatively homogeneous but differ in at least one distinguishing characteristic from beads in any other subset. Therefore, the subset to which a bead belongs can readily be determined after beads from different subsets are pooled.
In a preferred embodiment, the beads within each subset are uniform with respect to at least three and preferably four known classification parameter values measured with a flow cytometer: e.g., forward light scatter (C1) which generally correlates with size and refractive index; side light scatter (C2) which generally correlates with size; and fluorescent emission in at least one wavelength (C3), and preferably in two wavelengths (C3 and C4), which generally results from the presence of fluorochrome(s) in or on the beads. Because beads from different subsets differ in at least one of the above listed classification parameters, and the classification parameters for each subset are known, a bead""s subset identity can be verified during flow cytometric analysis of the pool in a single assay step and in real-time.
Prior to pooling subsets of beads to form a beadset, the beads within each subset can be coupled to a reactant that will specifically react with a given analyte of interest in a fluid sample to be tested. Usually, different subsets will be coupled to different reactants so as to detect different analytes. For example, subset 1 may be labeled so as to detect analyte A (AnA); subset 2 may be labeled so as to detect analyte B (AnB); etc.
At some point prior to assay, the variously labeled subsets are pooled. The pooled beads, or beadset, are then mixed with a fluid sample to test for analytes reactive with the various reactants bound to the beads. The system is designed so that reactions between the reactants on the bead surfaces and the corresponding analytes in the fluid sample will cause changes in the intensity of at least one additional fluorescent signal (Fm) emitted from a fluorochrome that fluoresces at a wavelength distinct from the wavelengths of classification parameters C3 or C4. The Fm signal serves as a xe2x80x9cmeasurement signal,xe2x80x9d that is, it indicates the extent to which the reactant on a given bead has undergone a reaction with its corresponding analyte. The Fm signal may result from the addition to the assay mixture of fluorescently labeled xe2x80x9csecondaryxe2x80x9d reagent that binds to the bead surface at the site where a reactant-analyte reaction has occurred.
When the mixture (pooled beads and fluid sample) is run through a flow cytometer, each bead is individually examined. The classification parameters, e.g., C1, C2, C3, and C4, are measured and used to classify each bead into the subset to which it belongs and, therefore, identify the analyte that the bead is designed to detect. The Fm value of the bead is determined to indicate the concentration of analyte of interest in the fluid sample. Not only are many beads from each subset rapidly evaluated in a single run, multiple subsets are evaluated in a single run. Thus, in a single-pass and in real-time a sample is evaluated for multiple analytes. Measured Fm values for all beads assayed and classified as belonging to a given subset may be averaged or otherwise manipulated statistically to give a single meaningful data point, displayed in histogram format to provide information about the distribution Of Fm values within the subset, or analyzed as a function of time to provide information about the rate of a reaction involving that analyte.
In a preferred embodiment, the beads will have two or more fluorochromes incorporated within or on them so that each of the beads in a given subset will possess at least four different classification parameters, e.g., C1, C2, C3, and C4. For example, the beads may be made to contain a red fluorochrome (C3), such as nile red, and bear an orange fluorochrome (C4), such as Cy3 or phycoerythrin. A third fluorochrome, such as fluorescein, may be used as a source of the Cn or Fm signal. As those of skill in the art will recognize, additional fluorochromes may be used to generate additional Cn signals. That is, given suitable fluorochromes and equipment, those of skill in the art may use multiple fluorochromes to measure a variety of Cn or Fm values, thus expanding the multiplexing power of the system even further.
In certain applications designed for more quantitative analysis of analyte concentrations or for kinetic studies, multiple subsets of beads may be coupled to the same reactant but at varying concentrations so as to produce subsets of beads varying in density of bound reactant rather than in the type of reactant. In such an embodiment, the reactant associated with classification parameter C4, for example, may be incorporated directly into the reactive reagent that is coupled to the beads, thereby allowing C4 conveniently to serve as an indicator of density of reactant on the bead surface as well as an indicator of reactant identity.
To prepare subsets varying in reactant density one may, for example, select, isolate, or prepare a starting panel of different subsets of beads, each subset differing from the other subsets in one or more of C1, C2, or C3. Each of those subsets may be further subdivided into a number of aliquots. Beads in each aliquot may be coupled with a reactant of choice that has been fluorescently labeled with a fluorochrome associated with C4 (e.g., Analyte A labeled with Cy3) under conditions such that the concentration or density of reactant bound to the beads of each aliquot will differ from that of each other aliquot in the subset. Alternatively, an entire subset may be treated with the C4 fluorochrome under conditions that produce a heterogeneous distribution of C4 reactant on beads within the subset. The subset may then be sorted with a cell sorter on the basis of the intensity of C4 to yield further subsets that differ from one another in C4 intensity.
One limitation of the alternative embodiment of using C4 labeled reactant as a classification agent is that one must design the system so that the value of C4 as a classification parameter is not lost. Therefore, one must take care to assure that the C4 intensities of all subsets carrying reagent A differs from the C4 intensities of all subsets carrying reagents B, C, and so forth. Otherwise, C4 would not be useful as a parameter to discriminate reactant A from reactant B, etc.
With either embodiment, the number of subsets that can be prepared and used in practice of the invention is theoretically quite high, but in practice will depend, inter alia, on the level of homogeneity within a subset and the precision of the measurements that are obtained with a flow cytometer. The intra-subset heterogeneity for a given parameter, e.g., forward angle light scatter C1, correlates inversely with the number of different subsets for that parameter that can be discriminated by flow cytometric assay. It is therefore desirable to prepare subsets so that the coefficients of variation for the value of each classification parameter (C1, C2, C3, and C4) to be used in a given analysis is minimized. Doing this will maximize the number of subsets that can be discriminated by the flow cytometer. Bead subsets may be subjected to flow cytometric sorting or other procedures at various different points in preparation or maintenance of the bead subsets to increase homogeneity within the subset. Of course, with simple assays designed to detect only a few different analytes, more heterogeneity can be allowed within a subset without compromising the reliability of the assay.
In an illustrative embodiment set forth here to explain one manner in which the invention can work in practice, the beads are used to test for a variety of antibodies in a fluid sample. A panel of bead subsets having known varying C1, C2, C3, and C4 values is first prepared or otherwise obtained. The beads within each subset are then coupled to a given antigen of interest. Each subset receives a different antigen. The subsets are then pooled to form an assay beadset and may be stored for later use and/or sold as a commercial test kit.
In the assay procedure, the beads are mixed with the fluid to be analyzed for antibodies reactive with the variety of antigens carried on the beads under conditions that will permit antigen-antibody interaction. The beads are labeled with a xe2x80x9csecondaryxe2x80x9d reagent that binds to antibodies bound to the antigens on the beads and that also bears the measurement fluorochrome associated with parameter Fm (e.g., fluorescein). A fluoresceinated antibody specific for immunoglobulin may be used for this purpose. The beads are then run through a flow cytometer, and each bead is classified by its characteristic classification parameters as belonging to subset-1, subset-2, etc. At the same time, the presence of antibodies specific for antigen A, B, etc., can be detected by measuring green fluorescence, Fm, of each bead. The classification parameters C1, C2, C3, and C4 allow one to determine the subset to which a bead belongs, which serves as an identifier for the antigen carried on the bead. The Fm value of the bead indicates the extent to which the antibody reactive with that antigen is present in the sample.
Although assays for antibodies were used above as an illustration, those of ordinary skill in the art will recognize that the invention is not so limited in scope, but is widely applicable to detecting any of a number of analytes in a sample of interest. For example, the methods described here may be used to detect enzymes or DNA or virtually any analyte detectable by virtue of a given physical or chemical reaction. A number of suitable assay procedures for detection and quantification of enzymes and DNA (particularly as the result of a PCR process) are described in more detail below.
The present invention also provides a significant advance in the art by providing a rapid and sensitive flow cytometric assay for analysis of genetic sequences that is widely applicable to detection of RNA, differing alleles, and any of a number of genetic abnormalities. In general, the methods of the present invention employ a competitive hybridization assay using DNA coupled microspheres and fluorescent DNA probes. Probes and microsphere-linked oligonucleotides could also include RNA, PNA, and non-natural nucleotide analogs.
In practice of the invention, oligonucleotides from a region of a gene of interest, often a polymorphic allele or a region to which a disease associated mutation has been mapped, are synthesized and coupled to a microsphere (bead) by standard techniques such as by carbodiimide coupling. A fluorescent oligonucleotide, complementary to the oligonucleotide on the bead, is also synthesized. To perform a test in accordance with the invention, DNA which is to be tested is purified and either assayed unamplified, or subjected to amplification by PCR, RT-PCR, or LCR amplification using standard techniques and PCR initiation probes directed to amplify the particular region of DNA of interest. The PCR product is then incubated with the beads under conditions sufficient to allow hybridization between the amplified DNA and the oligonucleotides present on the beads. A fluorescent DNA probe that is complementary to the oligonucleotide coupled to the beads is also added under competitive hybridization conditions. Aliquots of the beads so reacted are then run through a flow cytometer and the intensity of fluorescence on each bead is measured to detect the level of fluorescence which indicates the presence or absence of given sequences in the samples.
For example, when beads labeled with an oligonucleotide probe corresponding to a non-mutated (wild-type) DNA segment are hybridized with the PCR product from an individual who has a non-mutated wild-type DNA sequence in the genetic region of interest, the PCR product will effect a significant competitive displacement of fluorescent oligonucleotide probe from the beads and, therefore, cause a measurable decrease in fluorescence of the beads, e.g., as compared to a control reaction that did not receive PCR reaction product. If, on the other hand, a PCR product from an individual having a mutation in the region of interest is incubated with the beads bearing the wild-type probe, a significantly lesser degree of displacement and resulting decrease in intensity of fluorescence on the beads will be observed because the mutated PCR product will be a less effective competitor for binding to the oligonucleotide coupled to the bead than the perfectly complementary fluorescent wild-type probe. Alternatively, the beads may be coupled to an oligonucleotide corresponding to a mutation known to be associated with a particular disease and similar principles applied. In the multiplexed analysis of nucleic acid sequences, bead subsets are prepared with all known, or possible, variants of the sequence of interest and then mixed to form a bead set. The reactivity of the test sample, e.g. PCR product, with the wild-type sequence and other variants can then be assayed simultaneously. The relative reactivity of the PCR product with subsets bearing the wild-type or variant sequences identifies the sequence of the PCR product. The matrix of information derived from this type of competitive hybridization in which the test sequence and the entire panel of probe sequences react simultaneously allows identification of the PCR product as wild-type, known mutant, or unknown mutant. The invention thus provides one with the ability to measure any of a number of genetic variations including point mutations, insertions, deletions, inversions, and alleles in a simple, exquisitely sensitive, and efficient format.